) Ras, the product of the ras protoncogene, relays mitogenic signals from growth factor receptors and plays a prominent role in human carcinogenesis. Rho, a member of the greater Ras family, is necessary for Ras-induced transformation and plays an essential role in the processes of tumor cell invasion and metastasis. Both Ras and Rho are small G proteins which cycle between an active GTP-bound state and an inactive GDP-bound state. We developed a manual method to measure Ras activation in human tissue, i.e., determining the percentage of Ras molecules in the active GTP-bound state, and we recently modified this method to allow assessment of Rho I activation as well. Ras or Rho are isolated from tissue lysates and GTP and GDP bound to the proteins are eluted and quantitatively converted to ATP; ATP is measured by the firefly luciferase system which is sensitive to 1 fmol. We have found that Ras is highly activated in a significant number of breast, lung, and ovarian cancers, in the absence of a genetic ras mutation, with increased Ras activation correlating with increased expression of growth factor receptors. In a limited number of metastatic ovarian cancers, we have found Rho to be more highly activated in the metastatic lesion than in the primary tumor. Under the support of an R2l award, we developed a prototype instrument that allows full automated measurement of Ras and Rho activation (when combined with a commercially-available automated micro plate luminometer). We are now applying for an R33 award to test the instrument's accuracy, reproducibility, sensitivity, and specificity in measuring Ras and Rho activation in a series of breast, lung, and ovarian cancers. Results obtained with the instrument will be compared to results using the well-validated manual methods. Ras is one of the prime targets for cancer chemotherapy and a number of Ras inhibitors, some of which also inhibit Rho, have been developed and are in clinical trials. These drugs are most likely to be effective in tumors with increased Ras and/or Rho activation and, thus, measuring Ras/Rho activation in tumors will be of significant clinical relevance. On completion of the pilot testing phase proposed in these studies, the Automated Ras/Rho Activation Measurement Device should be ready for use in clinical laboratories.
